Introduction
The etiopathogenesis of systemic lupus erythe-matosus (SLE) is complex and still largely unknown. Genetic, environmental, and hormonal factors con-tribute to disease susceptibility (1). The importance of genetic factors is documented by 1) the pronounced difference in concordance rates between monozy-gotic and dizymonozy-gotic twins (15); 2) the higher disease prevalence in relatives of patients with SLE (14); 3) the
higher prevalence of SLE in certain ethnic groups, like African Americans and some Native Americans (18); and 4) studies of murine SLE showing that some strains invariably develop the disease (23, 24).
In humans, several genes contribute to lupus sus-ceptibility. Their identification has been complicated by the fact that SLE is a heterogeneous disease, both clinically and immunologically (3); in addition, many of the susceptibility genes differ across the various populations (27), and it is well recognized that genetic
MEDICINE®81: 169-78, 2002 Vol. 81, No. 3
Copyright © 2002 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A.
HLA Class II DNA Typing in a Large Series of European Patients
with Systemic Lupus Erythematosus
Correlations with Clinical and Autoantibody Subsets
MAUROGALEAZZI, GIANDOMENICOSEBASTIANI, GABRIELLAMOROZZI, CARLOCARCASSI, GIOVANNIBATTISTAFERRARA, RAFFAELLASCORZA, RICARDCERVERA, ENRIQUEDERAMONGARRIDO,
ANTONIOFERNANDEZ-NEBRO, FREDERICHOUSSIAU, ANNAJEDRYKA-GORAL, GIUSEPPEPASSIU,
CHRYSSAPAPASTERIADES, JEAN-CHARLESPIETTE, JOSEFSMOLEN, GIOVANNIPORCIELLO, ROBERTOMARCOLONGO,
AND THEEUROPEANCONCERTEDACTION ON THEIMMUNOGENETICS OFSLE*
169
From Istituto di Reumatologia (MG, GM, RM, GP), Università di Siena, Siena; Divisione di Reumatologia (GDS), Ospedale San Camillo, Roma; Dipartimento di Genetica Medica (CC), Istituto di Clinica Medica, and II Cattedra di Reumatologia (GP), Diparti-mento di Scienze Mediche, Centro Malattie Reumatiche Sis-temiche, Università di Cagliari, Cagliari; Laboratorio di Immuno-genetica (GBF), Istituto Nazionale per la Ricerca sul Cancro, Genova; Laboratorio di Immunologia Cellulare e Immunogenetica (RS), Istituto di Medicina Interna, Malattie Infettive e Immunopa-tologia, Università di Milano, Italy. Unitat de Malalties Autoim-munes Sistèmiques (RC), Hospital Clinic, Barcelona; Department of Medicine (EDRG), Lupus Unit, Universitary Hospital, Malaga; Unidad de Enfermedades Autoimmunes Sistemicas (AFN), Hospi-tal Regional del SAS, Malaga, Spain. Service du Rhumatologie (FH), Cliniques Universitaires Saint-Luc, Université Catholique de Lou-vain, Bruxelles, Belgium. Department of Connective Tissue Dis-eases (AJG), Institute of Rheumatology, Warsaw, Poland. Depart-ment of Immunology and Histocompatibility (CP), Evangelismos Hospital, Athens, Greece. Service de Médecine Interne (JCP), CHU Pitié-Salpetrière, Paris, France. 2nd Department of Medicine (JS), Center for Rheumatic Diseases, Lainz Hospital, Vienna, Austria.
This study was supported by a grant from the European Union on the Immunogenetics of SLE (European Concerted Action on Immunogenetics of SLE—Biomed 1; contract BMH1-CT94-1260).
*European Concerted Action on the Immunogenetics of SLE (ECAISLE): 1) Istituto di Reumatologia, Università di Siena,
Siena, Italy:M Galeazzi, R Marcolongo, G Morozzi, F Bellisai, G Porciello; 2) Divisione di Reumatologia, Ospedale San Camillo
de Lellis, Roma, Italy:GD Sebastiani, G Minisola; 3) II Cattedra
di Reumatologia, Dipartimento di Scienze Mediche, Centro Malattie Reumatiche Sistemiche, Università di Cagliari, Cagliari, Italy:A Mathieu, G Passiu, G Sanna, A Cauli; 4)
Labora-torio di Immunologia Cellulare e Immunogenetica, Istituto di
Medicina Interna, Malattie Infettive e Immunopatologia, Uni-versità di Milano IRCCS, Milano, Italy:R Scorza; 5)
Autoim-mune Diseases Research Unit, Servei de Medicina Interna Gen-eral, Hospital Clinic, Barcelona, Spain:J Font, R Cervera, M Ingelmo; 6) Service de Médecine Interne Godeau, CHU
Pitié-Salpetrière, Paris, France:J-C Piette; 7) Service de
Rhumatolo-gie, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Bruxelles, Belgium:F Houssiau; 8) Department of
Immunology and Histocompatibility, Evangelismos Hospital, Athens, Greece:C Papasteriades, K Boki, N Nicolopoulou; 9) 2nd
Department of Medicine, Center for Rheumatic Diseases, Lainz Hospital, Vienna, Austria:JS Smolen; 10) Servicio de Medicina
Interna & Section de Reumatologia, Hospital Clinico Universi-tario, Malaga, Spain:A Fenandez-Nebro, M de Haro-Liger, M Abarca-Costalago, J Rodriguez-Andreu; 11) Unidad de
Enfer-medades Autoinmunitarias Sistemicas, Servicio de Medicina Interna & Servicio de Nefrologia, Hospital Regional Carlos Haya, Malaga, Spain:E de Ramon Garrido, MT Camps Garcia, MA Frutos Sanz; 12) Department of Connective Tissue Diseases,
Institute of Rheumatology, University of Warsaw, Poland:A Jedryka-Goral, H Maldykowa, H Chwalinska-Sadowska; 13)
Di-partimento di Genetica Medica, Istituto di Clinica Medica, Uni-versità di Cagliari, Cagliari, Italy:L Contu, C Carcassi; 14)
Lab-oratorio di Immunogenetica, Centro di Biotecnologie Avanzate, Genova, Italy:GB Ferrara; 15) Dipartimento di Biologia
Moleco-lare, Unità di Immunochimica, Università di Siena, Siena, Italy:L Bracci; 16) Istituto di Scienze Neurologiche, Università
di Siena, Siena, Italy:P Annunziata. Other participants: Istituto
Dermopatico dell’Immacolata IDI-IRCCS, Roma, Italy:P Puddu, O De Pità, C Girardelli.
Address reprint requests to: Prof. Mauro Galeazzi, Institute of Rheumatology, Policlinico “Le Scotte,” Viale Bracci, 53100 Siena, Italy. Fax: 39-0-577-40450.
predisposition is linked to autoantibody repertoires and to clinical subsets of disease (9, 20, 31).
Many studies have focused on human leukocyte antigen (HLA) genes, mainly because they are in-volved in immune recognition and response, even if genetic susceptibility to SLE is in part located out-side the short arm of the sixth chromosome (13, 22, 30, 33, 35, 36). Studies performed by genomic scan of multiplex lupus families confirm the pathogenetic role of the HLA system in this disease (19).
We decided to approach the problem of HLA-SLE association in a large sample of patients, all of Cau-casian origin, to enable study of homogeneous sub-sets defined clinically and serologically. In this paper, we present the results of an immunogenetic study conducted in a large series of European SLE patients. Our objective was to determine the HLA class II al-lele association with SLE and its main clinical and au-toantibody subsets.
Patients and Methods
Five hundred seventy-seven patients with SLE (91.2% female, 8.8% male; female:male ratio 10:1), fulfilling 4 or more of the American Rheumatism Association (ARA) 1982 revised criteria for the classification of the disease (32), coming from 11 university hospitals of 7 European countries, were enrolled consecutively. Mean age of these patients was 37.9 13.1 years (range, 13–79 yr). Mean age at the onset of the disease was 29.5 12.5 years (range, 4–77 yr). Mean disease duration was 8.2 3.1 years (range, 1–24 yr). To avoid regional biases, each center was asked to contribute 60 patients, with the only exception the 2 centers in Malaga, in southern Spain, that were asked to give 30 patients each. Results were available for 577 patients, whose regional distribution is de-picted in Table 1.
Each of the centers recorded the clinical characteristics of the patients, including the past medical history and a complete physi-cal examination, using specific protocol forms, and collected blood and serum samples for the immunogenetic and serologic studies. All participating centers have substantial experience in
the management of patients with SLE. Nevertheless, in order to minimize the possible interobserver bias, the definition of the clin-ical features included in the protocol forms was discussed by all participating physicians on several occasions. (For a precise defi-nition of the clinical features, see references 5 and 29.)
Blood and serum samples were taken from patients at the time clinical information was collected and were stored at 80 C. Sub-sequently, they were transported in dry ice to the respective labo-ratories by the researchers of the clinical centers.
HLA class II typing was done at the following 3 laboratories, with the same methodology: 1) Department of Medical Genetics, Institute of Clinica Medica, University of Cagliari, Italy; 2) Im-munogenetics Laboratory, National Institute for Cancer Research, Genova, Italy; 3) Laboratory of Cellular Immunology and Immuno-genetics, Institute of Internal Medicine, Infectious Diseases and Im-munopathology, University of Milano, Italy. To guarantee quality control of the DNA typing tests and interlaboratory procedural uni-formity, there was exchange of personnel as well as of biologic ma-terial (blood samples, genetic probes) between the 3 laboratories where the tests were performed. In each patient, the following HLA loci were studied: DRB1, DRB3, DRB4, DRB5, DQA1, DQB1.
The frequency of HLA alleles in patients with SLE was compared with the frequency in a healthy Caucasian population, matched for ethnic origin, taken from the 12th International Histocompatibility Workshop (IHW) (11). Results of HLA class II allele typing of a healthy Polish population were from Jungerman et al (16).
Various serologic tests were performed, including the detection of autoantibodies that are more commonly found with SLE. Each serologic test was performed in the same laboratory for all pa-tients (Institute of Rheumatology, University of Siena, Italy). Cen-tralized data handling, computing, and statistical analysis took place at the Rheumatology Division, San Camillo Hospital, Rome, Italy. The data were transferred into a computerized database pro-gram (DBASE III PLUS) and were analyzed using the Statistical Package for Social Sciences (SPSS) program.
The study, approved by the Human Experimental Committee of each participating hospital, was carried out according to the prin-ciples of the Declaration of Helsinki. Informed consent was ob-tained from all patients.
Autoantibody detection
Antinuclear antibodies (ANA) were determined by indirect immunofluorescence using Hep-2 cells as substrate. Anti-dsDNA antibodies were determined by indirect immunofluorescence with Crithidia luciliae as substrate. Precipitating antibodies to extractable nuclear antigens, including Ro (SS-A), La (SS-B), U1-RNP, Sm, Jo1, SCL70, PCNA, were detected by double immuno-diffusion and counterimmunoelectrophoresis using calf and rab-bit thymus and human spleen extracts, as previously described (10, 20).
Other autoantibodies, such as anticardiolipin antibodies, anti-2glycoprotein I antibodies, antineutrophil cytoplasmic antibod-ies, and antiganglioside antibodantibod-ies, were also determined. Results of their correlation with clinical manifestation and HLA class II al-leles have been published separately (7–10).
HLA typing
Molecular typing of HLA-DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1 was performed according to the 11th IHW reference proto-col (17) with minor variations.
TABLE 1. Regional distribution of 577 European patients with systemic lupus erythematosus
Country/Region Center* No. of Patients
Belgium 7 60 France 6 60 Greece 8 58 Southern Spain 10 27 Southern Spain 11 37 Catalonia, Spain 5 60 Poland 12 60 Northern Italy 4 60 Central/southern Italy 2 60 Sardinia, Italy 3 60 Austria 9 35
*For the center reference number see list of the centers partic-ipating in this study (European Concerted Action on the Immuno-genetics of SLE) in footnote on first page of article.
DNA was extracted from peripheral blood mononuclear cells us-ing a standard phenol/chloroform technique. HLA-DRB, DQA1, DQB1 alleles were typed by the polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method, carried out us-ing digoxigenin (DIG)-labeled probes. The primers and the oligonu-cleotide probes used were those validated by the 11th IHW. Briefly, the second exon of each gene is specifically PCR amplified; the am-plified fragment is immobilized on a nylon positively-charged mem-brane (Zeta-Probe blotting memmem-brane, Bio-Rad, Hercules, CA) and hybridized with sequence-specific probes previously 3-end labeled with DIG-ddUTP using terminal-transferase (Boehringer Mannheim Gmbh, Germany). An anti-DIG-alkaline phosphatase conjugate (Fab fragments) (Boehringer Mannheim Gmbh) is bound to the DIG-labeled hybrids; incubation with CSPD (Tropix, Bedford, MA) sub-strate allows the chemiluminescent detection of the hybrids, by exposing the membranes to a X-Omat AR autoradiography film (Eastman Kodak, Rochester, NY).
Statistical analysis
Frequencies of HLA alleles were compared among the various groups using conventional chi-square analysis with the Yates cor-rection or, when appropriate, the Fisher exact test (2-tailed). The p value for HLA frequency differences was corrected (pc) for the number of comparisons. A p value equal to or less than 0.05 was taken as significant. An odds ratio (OR) was calculated, when the p value was significant, by using 22 contingency tables. When the value of a cell in the 22 table was 0, relative risk (RR) was cal-culated by Haldane’s modification of Woolf’s method (12):
RR
This statistical analysis was performed by means of the SPSS program using the information stored in the database program.
Results
Frequency of HLA class II alleles in patients and in healthy controls
As expected, frequency of HLA-DRB1*03 was sig-nificantly higher in SLE patients compared with healthy controls (35.2 vs 19.4, p 106, pc 1.3 105, OR 2.2, 95% CI [confidence intervals] 1.8–2.9) (Table 2). In addition, DRB1*15 and DRB1*16 (DR2) were significantly associated with SLE (re-spectively, 27.7 vs 19.4, p 0.003, pc 0.04, OR 1.6, 95% CI 1.2–2, and 14.4 vs 10.5, p 0.03, pc not significant [NS], OR 1.4, 95% CI 1.1–2). On the contrary, DRB1*01, DRB1*04, and DRB1*11 (DR5) showed negative association with SLE. Alleles at the DRB3 locus were not associated with the disease.
Among the alleles at the DQA1 locus, only DQA1*0102 showed a positive association with SLE (42.6 vs 32.8, p 2 104, pc 0.006, OR 1.5, 95% CI 1.2–1.9), whereas DQA1*0101 and DQA1*03 were negatively associated (Table 3).
Some alleles at the DQB1 locus were found with significantly increased frequency in patients with SLE compared with healthy controls. They were DQB1*0502 (17.8 vs 11.6, p 0.001, pc 0.03, OR 1.6, 95% CI 1.2–2.2), DQB1*0602 (20.7 vs 15.1, p 0.008, pc NS, OR 1.5, 95% CI 1.1–1.9), DQB1*0201 (52.7 vs 39.7, p 2 106, pc 5.6 105, OR 1.7, 95% CI 1.3–2.1), DQB1*0303 (5.8 vs 3.4, p 0.05, pc NS, OR 1.7, 95% CI 1–2.9), DQB1*0304 (1.9 vs 0.4, p 0.02, pc NS, OR 4.3, (d 1⁄ 2) (c 1 ⁄2) (a 1⁄ 2) (b 1 ⁄2)
TABLE 2. Phenotypic frequency of HLA-DRB1 and DRB3 alleles in patients with SLE and in healthy controls
Patients pc with SLE Controls p Value (p 13) **OR 95% CI
DRB1 (n 534) (n 958) *01 14.4 22.3 .003 .04 .6 .4–.8 *15 27.7 19.4 .003 .04 1.6 1.2–2 *16 14.4 10.5 .03 NS 1.4 1.1–2 *03 35.2 19.4 106 1.3 105 2.2 1.8–2.9 *04 14.6 21.3 .002 .03 .6 .5–.8 *11 20.2 27.3 .003 .04 .7 .5–.9 *12 3.4 1.9 NS *13 20.4 18.3 NS *14 5.4 6.3 NS *07 22.8 23.3 NS *08 5.6 5.3 NS *09 1.7 2 NS *10 2.2 2.3 NS DRB3 (n 376) (n 670) *01 44.2 38.2 NS *02 57.5 61.1 NS *03 11.2 15.3 NS
Abbreviations: SLE systemic lupus erythematosus; pc probability corrected for multiple comparison; OR odds ratio; CI confidence interval; NS not significant.
95% CI 1.2–16.2). Conversely, DQB1*0501, DQB1*0301, and DQB1*0302 showed a negative as-sociation with SLE (see Table 3).
Frequency of autoantibodies in lupus patients and correlation with HLA class II alleles
Frequencies of those autoantibodies of interest for this paper are depicted in Table 4. They are in keep-ing with the findkeep-ings in previous studies. With the ex-ception of anti-dsDNA (pc 0.001), the frequencies of all other autoantibodies were not statistically dif-ferent among the various centers. On the other hand, the frequency of anti-dsDNA in every patient was quite variable with time, depending mainly on the dis-ease activity and therapy.
As already known, anti-Ro was overrepresented in haplotypes containing DR3 (36.6 in patients DRB1*03 positive vs 19 in their negative counter-parts, p 2 105, pc 3 104, OR 2.4, 95% CI 1.6–3.7), as well as anti-La (13.1 vs 2.7, p 1 105, pc 1 104, OR 5.5, 95% CI 2.5–12.1)
(Table 5). Both these autoantibodies were also strongly associated with DQB1*0201 (DQw2), which is in strong linkage disequilibrium with DRB1*03, and, obviously, showed a strong association with haplotypes containing DRB1*03 and DQB1*0201. Anti-Ro, but not anti-La, was also associated with DQB1*0502 (DQw1), and with the haplotype DR2/
TABLE 3. Phenotypic frequency of HLA-DQA1 and DQB1 alleles in patients with SLE and in healthy controls
Patients pc with SLE Controls p Value (p 28) OR 95% CI
DQA1 (n 535) (n 900) *0101 17.2 29 106 2.8 105 .5 .4–.7 *0102 42.6 32.8 2 104 .006 1.5 1.2–1.9 *0103 12 14.2 NS *0104 4.9 .8 NS *0201 22.8 22.8 NS *03 16.6 23.7 .002 NS .6 .5–.8 *0401 5.6 4.6 NS *0501 53.5 50.6 NS *06 3.6 .6 NS DQB1 (n 535) (n 900) *0501 16.4 25 2 104 .006 .6 .4–.8 *0502 17.8 11.6 .001 .03 1.6 1.2–2.2 *0503 5 3.7 NS *0504 .4 .1 NS *0601 3.6 4.2 NS *0602 20.7 15.1 .008 NS 1.5 1.1–1.9 *0603 9.3 10.1 NS *0604 5.8 5.3 NS *0605 .4 .8 NS *0609 .9 .1 NS *0201 52.7 39.7 2 106 5.6 105 1.7 1.3–2.1 *0301 28.2 38.6 9 105 .002 .6 .5–.8 *0302 8.8 14.2 .003 NS .6 .4–.8 *0303 5.8 3.4 .047 NS 1.7 1–2.9 *0304 1.9 0.4 .02 NS 4.3 1.2–16.2 *0305 .2 .1 NS *0401 .9 1.2 NS *0402 4.3 4 NS *08 0.9 0.5 NS
Abbreviations: See previous tables.
TABLE 4. Relative frequency of autoantibodies in patients with SLE
Specificity Frequency dsDNA 56.2 Ro 25.2 La 6.3 Sm 7.2 U1-RNP 18.5 Jo1 0.2 SCL70 0.2 PCNA 0.9
Abbreviations: ds-DNA double-strand DNA; Sm Smith; U1-RNP U1-ribo-nuclear-proteins; SCL70 sclero 70; PCNA pro-liferating cells nuclear antigens.
DQB1*0502. It was not associated with DR2 (DRB1*15, DRB1*16). Anti-dsDNA was associated with DRB1*15 (66.2 vs 52.2, p 0.008, pc NS, OR 1.8, 95% CI 1.2–2.7), DQA1*0102 (63.6 vs 50.4, p 0.004, pc NS, OR 1.7, 95% CI 1.2–2.5), and DQB1*0602 (66 vs 53.5, p 0.03, pc NS, OR 1.7, 95% CI 1.1–2.7).
Anti-Ro showed negative association with DRB1*01 (11 vs 27.6, p 0.004, pc NS, OR 0.3, 95% CI 0.1–0.7), DRB1*14 (3.6 vs 26.5, p 0.01, pc NS, OR 0.1, 95% CI 0.01–0.8), DQA1*0101 (10.2 vs 28.3, p 6 104, pc 0.02, OR 0.3, 95% CI 0.1–0.6), DQB1*0501 (10.7 vs 28, p 0.001, pc 0.03, OR 0.3, 95% CI 0.1–0.6), and DQB1*0503 (0 vs 26.5, p 0.006, pc NS, OR 0.7, 95% CI 0.6–0.8). Anti-U1-RNP was negatively asso-ciated with DRB1*03 (12 vs 21.4, p 0.01, pc NS, OR 0.5, 95% CI 0.3–0.8), and DQA1*0501 (13 vs 24, p 0.002, pc NS, OR 0.5, 95% CI 0.3– 0.7). Anti-nDNA was negatively associated with DQB1*0301 (48.2 vs 59.1, p 0.04, pc NS, OR 0.6, 95% CI 0.4–0.9). The other autoantibodies showed no association with HLA class II alleles.
Correlation of clinical manifestations with HLA class II alleles and with autoantibodies
Frequency of clinical manifestation was compared among the 11 centers. The following clinical mani-festations showed a significantly different frequency: leukopenia (pc 6 104, ranging from 20% in pa-tients coming from Austria to 80% in papa-tients from Catalonia, Spain), lymphopenia (pc 6 104, rang-ing from 10% in patients from central/southern Italy to 85% in French patients), lymphadenopathy (pc 6 104, ranging from about 37% in French and Greek patients to about 20% in patients in all other centers), central nervous system involvement (pc 6 104, ranging from about 69% in patients from
France and Poland to 15% in those from northern Italy), livedo reticularis (pc 6 104, ranging from about 43% in patients from Poland and Greece to about 15% in those from the other centers). The as-sociation between these clinical manifestations and HLA alleles could be due to environmental factors or might be due to regional biases regarding diagno-sis, and has to be considered cautiously. All other clinical manifestations showed a similar frequency across the 11 centers.
Some HLA class II alleles were associated with clinical manifestations of SLE (Table 6), perhaps due to their association with autoantibodies. Indeed, DRB1*03 was associated with pleuritis, lung involve-ment, renal involveinvolve-ment, and psychosis. DRB1*16 carried a higher risk for renal involvement, discoid lupus, and lymphadenopathy. DQA1*0101 was asso-ciated with lymphadenopathy, and DQA1*03 with lung involvement, lymphopenia, and hemolytic ane-mia. DQB1*0201 (DQw2) was associated with leuko-penia, digital skin vasculitis, and pleuritis, while DQB1*0502 (DQw1) was associated with renal in-volvement, discoid lupus, and livedo reticularis.
Conversely, several HLA class II alleles were neg-atively associated with clinical manifestations of SLE (Table 7). DRB1*01 showed negative associations with renal and CNS involvement; DRB1*11 was neg-atively associated with renal disease and lymphope-nia; and DRB1*14 was negatively associated with leukopenia, lymphopenia, and digital skin vasculitis. Concerning the DQ locus, DQA1*0101 showed nega-tive associations with cardiac and renal disease, DQA1*0102 with CNS involvement, DQB1*0301 with arthritis and renal involvement, DQB1*0501 with re-nal involvement, and DQB1*0503 with leukopenia.
Some of these clinical manifestations were associ-ated with the presence of autoantibodies. Namely, anti-Ro was associated with pleuritis (39.9 in patients anti-Ro positive vs 22.8 in those anti-Ro negative, p
TABLE 5. Relative frequency of significantly associated autoantibodies in patients positive and negative for the various HLA alleles/haplotypes
HLA Anti-dsDNA Anti-Ro Anti-La p Value pc OR 95% CI
DRB1*03 36.6 vs 19 2 105 3 104 2.4 1.6–3.7 DRB1*03 13.1 vs 2.7 1 105 1 104 5.5 2.5–12.1 DRB1*15 66.2 vs 52.2 0.008 NS 1.8 1.2–2.7 DQA1*0102 63.6 vs 50.4 0.004 NS 1.7 1.2–2.5 DQA1*0501 8.7 vs 3.7 0.03 NS 2.5 1.1–5.4 DQA1*0601 55.6 vs 24.2 0.006 NS 3.9 1.5–10.2 DQB1*0201 33.5 vs 18.1 8 105 0.002 2.3 1.5–3.4 DQB1*0201 10.7 vs 2.5 3 104 0.008 4.6 2–10.9 DQB1*0502 37.4 vs 22.7 0.005 NS 2 1.3–3-3 DQB1*0602 66 vs 53.5 0.03 NS 1.7 1.1–2.7 DRB1*03/DQB1*0201 36.7 vs 19.2 2 105 3 104 2.4 1.6–3.7 DRB1*03/DQB1*0201 13.3 vs 2.7 1 105 1 104 5.6 2.6–12.4 DR2/DQB1*0502 37.6 vs 22.8 0.006 NS 2 1.2–3.3
1 104, OR 2.2, 95% CI 1.5–3-3), and with lung involvement (15.4 vs 5.2, p 2 10 4, OR 3.3, 95% CI 1.8–6.2). Anti-La was associated with pleuritis (44.4 vs 25.9, p 0.03, OR 2.3, 95% CI 1.15–4.5), and showed negative association with renal disease (2.8 vs 18.4, p 0.03, OR 0.1, 95% CI 0.02–0-9).
Although found in the context of SLE, other clini-cal manifestations that are more typiclini-cal of the anti-phospholipid syndrome are discussed in another paper (7).
Discussion
Multiple loci contribute to SLE, and disease patho-genesis is expressed as a complex genetic interaction that is undoubtedly influenced by environmental factors.
Genetic studies are complicated because the dis-ease is polygenic, with susceptibility derived from a number of genes with relatively small effects (30), possibly interacting (22). Genetic studies in SLE are
TABLE 6. Relative frequency of major clinical manifestations in patients positive and negative for the various HLA alleles (positive associations)
Clinical Manifestation HLA p Value OR 95% CI
Pleuritis DRB1*03 33.7 24.6 0.03 1.6 1–2.3 Lung involvement DRB1*03 12.5 6.1 0.02 2.2 1.2–4.1 Renal involvement DRB1*03 51.6 40.8 0.02 1.6 1.1–2.2 Psychosis DRB1*03 8.1 3.5 0.04 2.4 1.1–5.3 Raynaud DRB1*07 40.3 26.3 0.005 1.9 1.2–2.9 Renal involvement DRB1*16 26.3 16 0.04 1.9 1.1–3.3 Discoid lupus DRB1*16 25 12 0.004 2.4 1.3–4.4 Lymphadenopathy DRB1*16 26.3 13.8 0.009 2.2 1.2–3.8 Lymphadenopathy DQA1*0101 26.4 13.8 0.005 2.2 1.3–3.8 Raynaud DQA1*0201 39.5 26.8 0.01 1.8 1.2–2.8
Lung involvement DQA1*03 20 7.7 0.04 3 1.2–7.8
Lymphopenia DQA1*03 73.3 44.7 0.004 3.4 1.5–7.8
Hemolytic anemia DQA1*03 23.9 9.1 0.003 3.1 1.6–4.8
Leukopenia DQB1*0201 53.9 44.5 0.04 1.5 1–2.1 Digital vasculitis DQB1*0201 25.9 14.8 0.002 2 1.3–3.1 Pleuritis DQB1*0201 32.5 23.7 0.03 1.6 1.1–2.3 Lung involvement DQB1*0303 20 7.7 0.04 3 1.2–7.8 Lymphopenia DQB1*0302 61.7 44.9 0.04 2 1.1–3.7 Lymphopenia DQB1*0602 55.4 44 0.04 1.6 1–2.4 Renal involvement DQB1*0502 30.8 19.4 0.02 1.8 1.1–3 Discoid lupus DQB1*0502 24.5 11.6 0.002 2.5 1.4–4.3 Livedo reticularis DQB1*0502 31.9 21.3 0.04 1.7 1–2.8
Abbreviations: See previous tables.
TABLE 7. Relative frequency of major clinical manifestations in patients positive and negative for the various HLA alleles (negative associations)
Clinical Manifestation HLA p Value OR 95% CI
Renal involvement DRB1*01 10.5 23.3 0.02 0.4 0.2–0.8 CNS involvement DRB1*01 1.3 8.6 0.05 0.1 0.01–0.9 Renal involvement DRB1*11 28.3 43.9 0.005 0.5 0.3–0.8 Lymphopenia DRB1*11 34.9 49.2 0.01 0.5 0.4–0.9 Leukopenia DRB1*14 17.9 50.5 0.001 0.2 0.08–0.6 Lymphopenia DRB1*14 2.5 47.5 0.03 0.4 0.01–0.9 Digital vasculitis DRB1*14 3.6 20.8 0.05 0.1 0.02–0.9
Cardiac involvement DQA1*0101 2.2 10.1 0.03 0.2 0.05–0.8
Renal involvement DQA1*0101 13.2 23.2 0.05 0.5 0.3–0.9
CNS involvement DQA1*0102 39.4 48.8 0.04 0.7 0.5–0.9
Arthritis DQB1*0301 67.6 78.1 0.02 0.6 0.4–0.9
Renal involvement DQB1*0301 33.8 48.5 0.003 0.5 0.4–0.8
Renal involvement DQB1*0501 11.5 23.4 0.02 0.4 0.2–0.8
Leukopenia DQB1*0503 15.4 50.5 0.001 0.2 0.06–0.5
difficult also because SLE is heterogeneous, divisible in several clinical and immunologic subsets, each with its own genetic background. Furthermore, genes predisposing to SLE may vary according to the ethnic origin of the patient.
Studies, including association and family studies, have confirmed the linkage of the disease to the MHC region in humans (19). It also has been suggested that certain MHC haplotypes show stronger association with autoantibodies in SLE than with the disease it-self (2).
Studies of the association between HLA class II al-leles and SLE, and between HLA and serologic sub-types of the disease, were reviewed in depth by Reveille in 1999 (26), and we refer the reader to that paper for reference consultation. In Table 8 and Table 9 we have listed the most important associations de-scribed by other authors starting in 1998 (4, 6, 21, 25, 27, 28, 34). In summary, these studies found that HLA-DR3 was associated with Caucasian SLE patients in North America, the United Kingdom, Australia, Spain, Italy, Scandinavia, Switzerland, Austria, and, more re-cently, also in Germany (27) and the Netherlands (28), while in Greece the most significant association was with HLA-DR5 (DR11), and in northern India, with HLA-DR4. On the other hand the HLA-DR2 allele DRB1*1501 was associated with SLE in Caucasians and Asian groups including Japanese, Chinese, and Koreans, while in black patients the HLA-DR2 associ-ation was with the DRB1*1503 allele. In Mexican-Americans SLE was found to be associated with
HLA-DR3, and in both Mexican and Peruvian mesti-zos, with HLA-DR8. More recently (27) SLE was found to be associated with HLA-DQA1*0501 and DQB1*0201 in Caucasian patients and with HLA-DQB1*0602 in African Americans. In addition, in Mex-ican mestizo patients, an association has been found with HLA-DR3, DQA1*0501, DQB1*0201, and with HLA-DR1, DQA1*0101, and DQB1*0501 haplotypes (4). In Spanish patients an association of SLE with both HLA-DR3 and HLA-DQA1*0501 has been found (21), while in Greek SLE patients, associations with HLA-DRB1*1501, *1601, and *0701 have been de-scribed (34).
Concerning the HLA-DR and DQ genes and au-toantibody subsets of SLE, associations (26) have been found between anti-dsDNA antibodies and DR2 (DRB1*1501) and DR3 and their respective linked HLA-DQ alleles (DQA1*0102, DQB1*0602 and DQA1*0501, DQB1*0201), as well as, in those lacking these alleles, DQB1*0302. An association of anti-dsDNA antibodies with HLA-DR15 and Dw2 in French Canadian SLE patients has been reported (25).
Anti-Ro and anti-La antibodies have been found to be associated with HLA-DR2 and DR3 and with the concomitant presence of DQ1 and DQ2. Further-more, studies from 2 different European centers have implicated the HLA-B8-C4A*Q0-DRB1*0301-DQA1*0501-DQB1*0201 haplotype in susceptibility to anti-Ro/La autoantibody responses in Caucasians. In Japanese patients, the only association reported has been with HLA-DR8. The concomitant pro-duction of anti-Ro and anti-La antibodies has been found to be associated with HLA-DQA1*0501 and DQB1*0201 alleles in Spanish patients (21). More recently, an association has been found in Italy between anti-Ro/La response and HLA-DRB1*03011, DQA1*0501, and DQB1*0201 haplotype (6).
Anti-U1-RNP antibodies have been found to be as-sociated in both Japanese and Caucasians with HLA-DR4 and DQw3 (the serologic group comprising HLA-DQB1*0301, *0302, and *0303). Subsequent stud-ies, using oligotyping and DNA sequencing, have im-plicated both HLA-DRB1 and HLA-DQB1 alleles— specifically shared epitopes between HLA-DR2 and DR4 and HLA-DQB1*0302, linked to HLA-DR4—as
TABLE 8. HLA-DR, DQ associations with SLE in different ethnic groups
No. of
Ethnicity Patients HLA Associations Ref.
Netherlands 99 DRB1*03 28 Mexico 58 DR3, DQA1*0501, DQB1*0201 4 (Mestizo) DR1, DQA1*0101, DQB1*0501 Spain 85 DR3 21 DQA1*0501 Greece 46 DRB1*1501, DRB1* 1601 and 34 DRB1*0701 Haplotype.
TABLE 9. HLA class II alleles and autoantibody subsets of SLE
No. of
Autoantibody Ethnicity Patients HLA Associations Ref. Anti-ds DNA Canada 91 DR15 and Dw2 25
(French)
Anti-Ro/La Spain 85 DQA1*0501 and DQB1*0201 21 Anti-Ro/La Italy undefined DRB1*03011, DQA1*0501, DQB1*0201 6
well as other specific DQB1 alleles sharing specific epitopes with DQB1*0302 in both Caucasians and Japanese.
All studies mentioned above do have limitations. The studies often did not use homogeneous clinical criteria for patient assessment, and they used differ-ent laboratory methods for the detection of autoanti-bodies that cannot be compared. In addition the number of patients recruited is usually rather low.
Because of the above-mentioned considerations, we wanted to verify the characteristics of the associ-ation of SLE with HLA class II alleles in a large sam-ple of European patients. In fact, the large samsam-ple size allowed us to divide patients into various sub-sets, defined according to clinical characteristics and autoantibody status, and the common origin of the patients allowed us to overcome the problem of eth-nic influence on HLA-SLE association. On the other hand, results of our study may be hampered at least in part by the relatively high number of participating centers, because we can not rule out completely the influence of environmental factors and of biases re-garding diagnosis. The results presented here sug-gest that the overall contribution of the MHC to the disease itself is much less than its contribution to cer-tain types of autoantibodies (and, in turn, to related clinical manifestations).
In fact, we have already shown that particular HLA class II alleles (DRB1*04, DRB1*07, DQA1*0201, DQA1*0301, DQB1*0302), in the context of SLE, pre-dispose to the production of antiphospholipid anti-bodies and, as a consequence, to the development of clinical manifestations related to the antiphospho-lipid syndrome (9). We have also found significant association between other HLA alleles and both anti-ganglioside antibodies and related clinical manifes-tations (7).
In this paper, we show that, in the same patient sam-ple, other HLA class II alleles confer susceptibility for different autoantibodies and possibly related clinical manifestations. For instance, patients carrying the DRB1*03 allele and the closely linked DQB1*0201 al-lele show genetic predisposition to the production of autoantibodies to Ro, La, and to pulmonary involve-ment, pleuritis, and psychosis. Interestingly, a similar association between DQB1*0201 and Ro/La anti-bodies has been previously described in a Spanish population. To our knowledge, we have found for the first time that patients carrying the DQB1*0502 allele, which is in linkage disequilibrium with DR2, are prone to develop anti-Ro antibodies without anti-La antibodies, renal disease, discoid lupus, and livedo reticularis, whereas the presence of anti-La antibod-ies might be protective for renal disease. The associ-ation of Raynaud phenomenon and DRB1*07 (never described before, to our knowledge) is probably a consequence of the association of this allele with
antiphospholipid antibodies (9). DR2 (DRB1*15, DRB1*16) was associated with anti-dsDNA antibod-ies, confirming data previously described in Cau-casian populations. It is noteworthy that we found for the first time, as far as we know, that DR2 haplo-types were also associated with renal disease, dis-coid lupus, and lymphadenopathy. DRB1*11 (DR5) appeared to protect against severe lupus, character-ized by nephritis and lymphopenia, while SLE pa-tients carrying DR1 showed a decreased risk for renal and CNS involvement, together with a de-creased frequency of anti-Ro. Furthermore, DRB1*14 was associated with a decreased risk of having anti-Ro, and patients with this allele were less prone to develop digital skin vasculitis, leukopenia, and lym-phopenia. The negative association of anti-dsDNA antibodies with DQB1*0301 could explain why pa-tients with this allele had less renal disease.
Therefore, our results confirm that the role of HLA class II genes in SLE is to influence the production of the autoantibody repertoire and clinical manifesta-tions, whereas the onset of SLE is more likely the consequence of the cooperation of many other non-HLA genes, interacting with appropriate external stimuli. Indeed, recent family studies showed that the genetic predisposition to SLE is only in minimal part located on the short arm of the sixth chromo-some. Other regions of the human genome appear to be more important for SLE susceptibility, such as those located in chromosome 1, 2, 14, 16, and 20 (19). In addition, family studies show that different sus-ceptibility loci are involved in the development of SLE in different ethnic populations, confirming the genetic heterogeneity of this disease.
In conclusion, our study of a large sample of Cau-casian patients with lupus shows some new HLA clin-ical and serologic associations in SLE and reinforces the concept that this disease is genetically complex and that the role of MHC genes is mainly to predispose to particular serologic and clinical manifestations.
Summary
We conducted this study to determine the HLA class II allele associations in a large cohort of pa-tients of homogeneous ethnic derivation with sys-temic lupus erythematosus (SLE). The large sample size allowed us to stratify patients according to their clinical and serologic characteristics. We studied 577 European Caucasian patients with SLE. Antinuclear antibodies (Hep-2 cells), anti-dsDNA antibodies (Crithidia luciliae), and antibodies to extractable nuclear antigens Ro (SS-A), La (SS-B), U1-RNP, Sm, Jo1, SCL70, and PCNA, were detected in all patients. Molecular typing of HLA-DRB1, DRB3, DQA1, and DQB1 loci was performed by the polymerase chain
reaction-sequence specific oligonucleotide probes (PCR-SSOP) method.
We found a significantly increased frequency of DRB1*03, DRB1*15, DRB1*16, DQA1*0102, DQB1*0502, DQB1*0602, DQB1*0201, DQB1*0303, and DQB1*0304 in lupus patients as compared with healthy controls. In addition, DRB1*03 was associ-ated with anti-Ro, anti-La, pleuritis, and involvement of lung, kidney, and central nervous system. DRB1*15 and DQB1*0602 were associated with dsDNA antibodies; DQB1*0201 with Ro and anti-La, leukopenia, digital skin vasculitis, and pleuritis; and DQB1*0502 was associated with anti-Ro, renal involvement, discoid lupus, and livedo reticularis.
In conclusion, our study shows some new HLA clinical and serologic associations in SLE and further confirms that the role of MHC genes is mainly to pre-dispose to particular serologic and clinical manifes-tations of this disease.
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